Abstract 377: Correction of VLCAD Deficiency in Mice: Steps Toward a Gene Therapy for Cardiomyopathy
Cardiomyopathy is an important cause of morbidity and mortality in children. Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD), one of several nuclear encoded mitochondrial enzymes that catalyze the initial step in the beta-oxidation of straight chain fatty acids, often presents with cardiomyopathy and/or sudden death. How VLCAD deficiency causes cardiomyopathy is unclear, but appears to be related to the accumulation of toxic long-chain acyl-carnitine species rather than a block in energy metabolism. While dietary therapy for VLCAD deficiency has been beneficial to some, many people do not respond. Intercurrent illness with diminished oral intake, exercise, or fasting all increase demand for fatty acid metabolism and can potentially trigger catastrophic events even while on dietary therapy. A murine knock-out model of VLCAD deficiency is available and shows a milder disease than that seen in humans, possibly due to differences in the substrate specificity and/or tissue distribution of VLCAD between humans and mice. Here we report here the development of a recombinant AAV (pseudotype 8) vector that expresses the human VLCAD gene (AAV8-hVLCAD). In vivo studies monitoring long chain acyl-carnitine levels in the serum of treated and control mice following IV injection of 3 × 1011 AAV8-hVLCAD vector genomes shows a reduction in long chain acyl-carnitine species to levels similar to those seen in wild-type litter mates within 1–2 weeks. Correction of fasting hypoglycemia is also seen in treated mice compared to untreated controls. Western analysis of liver, skeletal muscle, and heart extracts using the novel Pep-1 anti-VLCAD antibody demonstrate hVLCAD expression in the liver, and almost no expression in the skeletal muscle and heart. Finally, in vitro studies with a luciferase report construct identifies a minimal VLCAD promoter that functions well in human cells. This work demonstrates the short term correction of serologic abnormalities seem in the VLCAD deficient mice, and identifies a native VLCAD promoter fragment for use in future long-term in vivo expression studies.